Angiogenesis has emerged as attractive therapeutic target due to its implication in a variety of pathological conditions, including tumor growth, proliferative retinopathies, age-related macular degeneration, rheumatoid arthritis (RA), and psoriasis (Folkman et al., 1992, J. Biol. Chem. 267:10931-10934). The first indication of specific molecular angiogenic factors was based on the observation of the strong neovascular response induced by transplanted tumors. It is now known that angiogenesis is essential for the growth of most primary tumors and their subsequent metastasis. Numerous molecules have since been associated with the positive regulation of angiogenesis, including transforming growth factor (TGF)-α, TGF-β, hepatocyte growth factor (HGF), tumor necrosis factor-α, angiogenin, interleukin (IL)-8, and vascular endothelial growth factor (VEGF, also referred to as VEGFA or vascular permeability factor (VPF)) (Ferrara et al., 2003, Nature Medicine 9:669-676).
The VEGF proteins are important signaling proteins involved in both normal embryonic vasculogenesis (the de novo formation of the embryonic circulatory system) and abnormal angiogenesis (the growth of blood vessels from pre-existing vasculature) (Ferrara et al., 1996, Nature 380:439-442; Dvorak et al., 1995, Am. J. Pathol. 146:1029-1039). VEGF is associated with solid tumors and hematologic malignancies, interocular neovascular syndromes, inflammation and brain edema, and pathology of the female reproductive tract (Ferrara et al., 2003, Nature Medicine 9:669-676). VEGF mRNA is over-expressed in many human tumors, including those of the lung, breast, gastrointestinal tract, kidney, pancreas, and ovary (Berkman et al., 1993, J. Clin. Invest. 91:153-159). Increases in VEGF in the aqueous and vitreous humor of the eyes have been associated with various retinopathies (Aiello et al., 1994, N. Engl. J. Med. 331:1480-1487). Age-related macular degeneration (AMD), a major cause of vision loss in the elderly is due to neovascularization and vascular leakage. The localization of VEGF in the choroidal neovascular membranes in patients affected by AMD has been shown (Lopez et al., 1996, Invest. Ophtalmo. Vis. Sci. 37:855-868).
The VEGF gene family includes the prototypical member VEGFA, as well as VEGFB, VEGFC, VEGFD, and placental growth factor (PLGF). The human VEGFA gene is organized as eight exons separated by seven introns. At least six different isoforms of VEGF exist, VEGF121, VEGF145, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189, and VEGF206, where the subscripts refer to the number of amino acids remaining after signal cleavage. Native VEGF is a 45 kDa homodimeric heparin-binding glycoprotein (Ferrara et al., 2003, Nature Medicine 9:669-676). VEGF (specifically VEGFA) binds to two related receptor tyrosine kinases, VEGFR-1 (also referred to as Flt-1) and VEGFR-2 (also referred to as Flk-1 or kinase domain region (KDR) or CD309). Each receptor has seven extracellular and one transmembrane region. VEGF also binds to the neuropilins NRP1 (also referred to as vascular endothelial cell growth factor 165 receptor (VEGF165R) or CD304) and NRP2 also referred to as vascular endothelial cell growth factor 165 receptor 2 (VEGF165R2)).
Given its central role in regulating angiogenesis, VEGF provides an attractive target for therapeutic intervention. Indeed, a variety of therapeutic strategies aimed at blocking VEGF or its receptor signaling system are currently being developed for the treatment of neoplastic diseases. The anti-VEGF antibody bevacizumab, also referred to as rhuMAb VEGF or Avastin®, is a recombinant humanized anti-VEGF monoclonal antibody created and marketed by Genentech (Presta et al., 1997, Cancer Res. 57:4593-4599). In order to construct bevacizumab the complementarity-determining regions (CDRs) of the murine anti-VEGF monoclonal antibody A.4.6.1 were grafted onto human frameworks and an IgG constant region. Additional mutations outside the CDRs were then introduced into the molecule to improve binding, affording an antibody in which ˜93% of the amino acid sequence is derived from human IgG1 and ˜7% of the sequence is derived from the murine antibody A.4.6.1. Bevacizumab has a molecular mass of about 149,000 Daltons and is glycosylated.
Ranibizumab is an affinity maturated Fab fragment derived from bevacizumab. Ranibizumab has a higher affinity for VEGF and also is smaller in size, allowing it to better penetrate the retina, and thus treat the ocular neovascularization associated with AMD (Lien and Lowman, In: Chemajovsky, 2008, Therapeutic Antibodies. Handbook of Experimental Pharmacology 181, Springer-Verlag, Berlin Heidelberg 131-150). Ranibizumab was developed and is marketed by Genentech under the trade name Lucentis®.
Treatment of cancer patients with a regimen that includes Avastin® can result in side effects including hypertension, proteinuria, thromboembolic events, bleeding and cardiac toxicity (Blowers & Hall, 2009, Br. J. Nurs. 18(6):351-6, 358). Also, despite being a humanized antibody, bevacizumab can elicit an immune response when administered to humans. Such an immune response may result in an immune complex-mediated clearance of the antibodies or fragments from the circulation, and make repeated administration unsuitable for therapy, thereby reducing the therapeutic benefit to the patient and limiting the re-administration of the antibody.
Accordingly, there is a need to provide improved anti-VEGF antibodies or fragments that overcome one or more of these problems, for example, by generating variants with higher affinity than bevacizumab that can be administered at reduced dosages, or variants with reduced immunogenicity and other side-effects as compared to bevacizumab.
Citation or identification of any reference in Section 3 or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present disclosure.